‘84K’杨组氨酸激酶基因PaHK3b的克隆及功能分析

Cloning and Functional Analysis of Histidine Kinase Gene PaHK3b in Poplar'84K'

[目的]本研究克隆了银腺杨‘84K’(Populus alba×P.glandulosa‘84K’)组氨酸激酶基因PaHK3b启动子及编码区,并对其表达进行检测及功能鉴定,为深入研究PaHK3b基因在杨树生长发育的调控作用提供线索,为杨树分子育种及品种改良奠定基础。[方法]根据毛果杨(P.trichocarpa Torr.&Gray)基因组信息,设计引物克隆‘84K’杨组氨酸激酶基因PaHK3b启动子及CDS序列,并对其保守结构域和启动子顺式作用元件进行分析;同时,对‘84K’杨进行植物激素处理(10μmol·L^(−1)ABA、10μmol·L^(−1)6-BA、10μmol·L^(−1) IBA、10μmol·L^(−1) GA3及10μmol·L^(−1)水杨酸(SA))及非生物胁迫处理(42℃高温、0℃低温、200 mmol·L^(−1) NaCl和5%PEG6000),利用实时定量PCR(qRT-PCR)方法检测PaHK3b基因表达情况与表达响应差异,并采用原核表达方法初步确定PaHK3b基因的生物学功能。[结果]PaHK3b基因编码框区长度为3060 bp,编码1019个氨基酸,PaHK3b蛋白具有CHASE、HisKA和REC等典型的细胞分裂素受体结构域。PaHK3b基因启动子序列中不仅含有大量TATA框和CAAT框常见核心元件,还包含低温响应元件LTR、防御与胁迫响应元件TC-rich repeats、赤霉素响应元件GARE-motif、水杨酸响应元件TCA-element等顺式作用元件,这些元件与杨树的激素响应和逆境胁迫响应密切相关。qRT-PCR分析表明:PaHK3b基因在叶片中表达最高,根部中等,茎中最少;另外,与正常条件下相比,在高温、低温、NaCl及PEG处理时,PaHK3b基因表达量与对照明显增高,分别为对照的2.67、2.61、2.28、1.87倍;用IBA诱导处理时,基因表达量与对照相比差异不大,而在6-BA、ABA、GA3及SA处理时,基因表达量与对照相比均呈下调表达;在添加5%PEG6000的LB液体培养基中,转入PaHK3b基因原核表达载体的大肠杆菌菌株生长速度显著高于对照,在添加50~150 mmol·L^(−1)NaCl的LB固体培养基上,转入PaHK3b基因原核表达载体的大肠杆菌菌株单克隆生长均好于对照。[结论]‘84K’杨PaHK3b基因启动子含有逆境和激素响应元件,表明PaHK3b基因与杨树植物激素信号及非生物胁迫信号响应密切相关。经非生物胁迫处理、激素处理及原核表达证实,杨树PaHK3b基因参与杨树植物激素信号响应,并在其抗逆境胁迫过程中发挥重要调控作用。

[Objective]Using Poplar'84K'(Populus alba×P.glandulosa)as material to clone the promoter region and coding sequence(CDS)of histidine kinase gene PaHK3b,then to detect its expression and identify its functions,so as to provide clues for analyzing the function of PaHK3b in growth and development regulation of poplars and for molecular breeding and genetic improvement of poplar.[Method]Based on the published genome information of P.trichocarpa Torr.&Gray,the promoter region and CDS were cloned using the specific primers designed.The conservative structure domain and promoter cis-acting element were analyzed.At the same time,in vitro plants of Poplar'84K'were treated with several plant hormones(10μmol·L^(−1) ABA,10μmol·L^(−1)6-BA,10μmol·L^(−1) IBA,10μmol·L^(−1) GA3,and 10μmol·L^(−1) SA)under various abiotic stresses(42℃,0℃,200 mmol·L^(−1) NaCl,and 5%PEG6000).Differences in expression and response of PaHK3b gene were detected.Furthermore,the prokaryotic expression system was used to study the biological function of PaHK3b gene in vitro.[Result]The PaHK3b gene was 3060 bp in length and encoded 1019 amino acids.PaHK3b protein had a typical cytokinin receptor domain,a CHASE domain,a HisKA domain,and a REC domain.Besides,a large number of common core elements of TATA box and CAAT box,a number of stress-related and hormone-related regulatory elements,such as low temperature response element LTR,defense and stress responsive element TC-rich repeats,gibberellin responsive element GAREmotif,salicylic acid responsive element TCA-element etc.were predicted in the promoter sequence of PaHK3b gene,that closely related to the plant hormone and stress response regulations.The qRT-PCR results showed that PaHK3b gene was expressed the highest in leaf,medium in root,and least in stem.The transcriptional levels of PaHK3b gene were about 2.67,2.61,2.28,1.87 times that of the control respectively under 42℃,0℃,200 mmol·L^(−1) NaCl,and 5%PEG treatments.Under IBA treatment,the transcripts of PaHK3b were not significantly different from that of the control.The expression of PaHK3b were down-regulated under the treatments of 6-BA,ABA,GA3 and SA respectively.The growth of the Escherichia coli strain with PaHK3b prokaryotic expression construction in LB liquid medium supplemented with 5%PEG6000 were faster than that of the control.Meanwhile,the growth of the E.coli strain with PaHK3b prokaryotic expression construction on solid LB contained 50~150 mmolL^(−1) NaCl was better than that of the control.[Conclusion]The promoter of poplar'84K'PaHK3b gene contained cis-regulatory elements,such as stress response element and hormone responsive element etc,showing that PaHK3b gene is closely related to the response of poplar to hormones and abiotic stress.It is confirmed by abiotic stress treatment,hormone treatment and prokaryotic expression that PaHK3b gene participates in the signal response of plant hormones and plays an important role in poplar response to abiotic stresses.